Abstract 18259: Inhibition of STIM1 Oligomerization by Protein O-Linked N-Acetylglucosamine: A Novel Metabolically Mediated Regulation of Cardiomyocyte Calcium Overload
We have previously shown that acute increases in the levels of O-linked N-acetylglucosamine (O-GlcNAc) are cardioprotective, in part by attenuating Ca2+ mediated injury, and also that increased O-GlcNAcylation blunts non-voltage gated store-operated calcium entry (SOCE) in cardiomyocytes. In non-excitable cells, stromal interaction molecule 1 (STIM1) is the primary ER calcium sensor facilitating SOCE. Therefore, we tested the hypothesis that O-GlcNAc cardioprotection is due, in part, to inhibition of STIM1-mediated SOCE. Using immunoblot and immunohistochemistry we showed that STIM1 was present in neonatal rat ventricular myocytes (NRVM) and the adult rat heart. In the heart STIM1 co-localized with SERCA consistent with ER/SR localization. NRVM were transfected with eYFP-tagged STIM1; under basal conditions STIM1 occurred in a distributed reticular network. ER/SR Ca2+ depletion induced by 2mM EGTA and 5μM thapsigargin (TG) resulted in the rapid formation of STIM1 puncta, the required first step in STIM1-mediated SOCE. Increasing NRVM O-GlcNAc levels with either glucosamine (GlcN, 5mM) or thiamet-G (10μM), which stimulate O-GlcNAc synthesis and inhibit its degradation, respectively, significantly (p<0.05) attenuated the % of STIM1 puncta-positive cells induced by Ca2+ depletion. Immunoprecipitation studies followed by anti-O-GlcNAc immunoblot indicate preliminarily that STIM1 is itself a target for O-GlcNAc modification. Pretreatment of adult cardiomyocytes with GlcN significantly attenuated cell death induced by addition of Ca2+ following TG treatment, as predicted by our earlier finding that GlcN protects the intact heart against calcium paradox-induced Ca2+ overload. These data suggest therefore that STIM1-mediated SOCE is an important contributor to the Ca2+ entry that results in Ca2+ overload and cell death following SR Ca2+ depletion. Further, these data and the finding by others that SR Ca2+ depletion occurs upon reperfusion, provide a mechanistic explanation for our previous demonstration that increased O-GlcNAc mediates cardioprotection following ischemia. This leads to the provocative hypothesis that a novel STIM1-mediated SOCE pathway is an important contributor to reperfusion injury in the heart.
- © 2010 by American Heart Association, Inc.